Drugs for the treatment of sarcoglycanopathies

ABSTRACT

Inhibitors of the endoplasmic reticulum associated degradation (ERAD) pathway, particularly inhibitors of mannosidase I, are used for the preparation of a medicinal product intended to treat sarcoglycanopathies.

TECHNICAL FIELD

This invention relates to the treatment of sarcoglycanopathies.

More precisely, the invention concerns the use of inhibitors of theendoplasmic reticulum associated degradation pathway, particularlymannosidase I inhibitors, as medicinal products intended for thetreatment of certain forms of the disease.

PRIOR ART

Sarcoglycanopathies are autosomal recessive muscular disorders of theLimb Girdle Muscular Dystrophy (LGMD) group. Four forms of the diseasehave been identified (LGMD 2C, LGMD 2D, LGMD2E, and LGMD 2F), resultingrespectively from defects in the γ-, α-, β- and δ-sarcoglycan genes (11,17).

LGMD2C is particularly widespread in the Mediterranean basin and inGypsy populations residing in Europe. LGMD2D has been diagnosed acrossthe world in patients in Europe, Africa, Japan and Brazil. LGMD2E hasbeen diagnosed for the most part in the Amish community and in NorthAfrica. Until now, LGMD2F has only been reported in 7 families ofBrazilian, Turkish and Italian origin.

Patients with sarcoglycanopathies present progressive muscular weaknessof the pelvic and shoulder girdle muscles which is often associated withcalf hypertrophy. Cognitive impairment is absent. Cardiac complicationssuch as cardiomyopathies are sometimes observed in LGMD2C, LGMD2E andLGMD2F patients.

Upon histological examination, muscles show areas of regeneration anddegeneration, inflammatory infiltrates, fibrosis and variation in fibresize. The severity of the disease is variable, even among siblings. Inthe most severe cases, patients lose the ability to walk before the ageof 30 and their life expectancy is reduced.

Recurrent mutations occur in two forms of the disease. In LGMD2Cpatients, the de1521T and C283Y mutations are frequently observed inNorth African and Gypsy populations, respectively (4, 13).

The most frequent mutation found in LGMD2D patients is the R77Csubstitution where the arginine in position 77 is replaced by acysteine. One third of European patients carry this mutation, except inFinland where it is observed in 100% of patients, most often on bothalleles (5, 9).

Sarcoglycans (SG) are a group of plasma membrane glycoproteins thatassociate with dystrophin to form the dystrophin glycoprotein complex(12). This complex contributes to the mechanical link between the actincytoskeleton and the extracellular matrix and consequently plays a rolein muscle fibre membrane stability (10). Sarcoglycans consist of a smallintracellular domain that can be either C-terminal (α-sarcoglycan) orN-terminal (γ-, β- and δ-sarcoglycans), a single transmembrane domainand a large extracellular domain bearing N-glycosylation sites.

Sarcoglycan complex assembly occurs during transport of these proteinsin the sarcoplasmic reticulum and the Golgi apparatus (6, 16). Assemblyis initiated by β-sarcoglycan which binds to δ-sarcoglycan to positionthe complex correctly within the membrane. Then, α-sarcoglycan binds tothe complex and in turn binds γ-sarcoglycan for complete assembly.Mutations in any sarcoglycan can disrupt assembly of the complex,causing secondary deficiency of the other sarcoglycans.

Whilst today we know how to diagnose sarcoglycanopathies, and evenattribute the phenotype to specific mutations in the γ-, α-, β- orδ-sarcoglycans, we still have few options for treating patients withthis disease.

Until now, no specific treatment has been developed and most patientsrequire physiotherapy to avoid muscle cramps worsening. However, severalteams have reported positive results in experiments using gene transfervia viral vectors or cell therapy in animal models (1, 7, 14). For thisreason, document WO 00/20582 recommends use of a gene therapy approachto replace the defective sarcoglycan gene, in particular using AAVvectors.

In the prior art, an overall therapeutic approach, in which proteasomefunction was inhibited, was put forward for the treatment of musculardisorders, in particular muscular dystrophies. However, thisnon-specific approach does not seem to be particularly effective andcould lead to tolerance problems.

It is therefore critical to develop new therapeutic approaches, inparticular with new medicinal products that specifically and effectivelytreat sarcoglycanopathy patients

OBJECT OF THE INVENTION

This invention is based on results reported by the inventorsdemonstrating that:

-   -   some sarcoglycan mutations associated with sarcoglycanopathies        produce glycoproteins displaying defective folding;    -   these misfolded proteins are disposed of via the endoplasmic        reticulum associated glycoprotein degradation pathway;    -   inhibitors of this pathway block defective sarcoglycan protein        degradation;    -   if not degraded (in the presence of inhibitors), defective        sarcoglycans can be correctly translocated to the plasma        membrane and assembled in the sarcoglycan complex, leading to        recovery of a normal phenotype.

It should be recalled that endoplasmic reticulum (ER) glycosidases playan important role in controlling the quality of glycoprotein production.They ensure that only correctly folded glycoproteins are transported totheir final location. In particular, cleavage of mannose residues in theER by α-mannosidases acts as a signal guiding misfolded glycoproteinstowards the proteasome for degradation.

α-mannosidases belong to two groups. Class I mannosidases (mannosidaseI) are inhibited by 1-deoxymannojirimycin and kifunensin, while class IImannosidases are specifically inhibited by swainsonine. The proteasomeis a protein complex involved in the degradation of intracellularproteins in the cytosol and can for example be inhibited by MG132 orbortezomib.

In theory any inhibitor of this degradation pathway (ERAD: EndoplasmicReticulum Associated Degradation) could be used. However, this inventionhas focused on mannosidase I inhibitors which have proved to have a veryselective mode of action and are remarkably efficient for treatingsarcoglycanopathies. From a clinical point of view, this notablycorresponds to potentially lesser adverse effects.

Consequently, the invention relates to the use of class I α-mannosidaseinhibitors for the preparation of a medicinal product intended to treatsarcoglycanopathies.

It is therefore clear that in the context of this invention, use of atleast one mannosidase I inhibitor has been investigated. Naturally, theuse of a “cocktail” of inhibitors is not rejected, namely severalinhibitors of different types, with complementary inhibitory activities,particularly in regard to their activity/selectivity

Kifunensin (CAS 109944-15-2) and 1-deoxymannojirimycin (CAS 84444-90-6)molecules are particularly preferred since they are known to inhibitmannosidase I.

However, the scope of this invention is not limited to these molecules.Indeed, any candidate molecule may be tested for its mannosidase Iinhibitory activity using a simple enzymatic assay.

Considering that the aim of this approach is to find therapeuticsolutions for human sarcoglycanopathies, the enzyme used for testingshould be a class 1 alpha-mannosidase of human origin, particularly theenzyme referenced in databases under accession number: NP_(—)005898(mannosidase, alpha, class 1A, member 1 [Homo sapiens]).

An enzymatic assay suitable for testing such inhibitors was, forexample, described in the article published in J. Biol. Chem. 2004 Oct.8; 279(41):42638-47. Epub 2004 Jul. 22. “The twisted abdomen phenotypeof Drosophila POMT1 and POMT2 mutants coincides with their heterophilicprotein O-mannosyltransferase activity”, Ichimiya T, Manya H, Ohmae Y,Yoshida H, Takahashi K, Ueda R, Endo T, Nishihara S.

The method of administration, the dose administered, and the frequencyof administration are determined for each specific case, using classicprotocols known to those skilled in the art. These parameters dependparticularly on the mutation to be treated and the inhibitor used.

Kifunensin is a preferable choice. This inhibitor is highly specific andhas the advantage of being water-soluble and is therefore suitable fororal administration.

Besides, this method of treatment is particularly appropriate fortreating sarcoglycanopathies caused by the human α-sarcoglycan R77Cmutation (Arg77Cys in the protein corresponding to a 229C>T mutation onthe gene). As previously discussed, this mutation is responsible fornumerous clinical cases, especially in Europe.

However, the applicant has demonstrated that 1-deoxymannojirimycin canalso be used instead of kifunensin.

In addition, in the context of this invention, it has been demonstratedthat the effect observed is not specific to a particular mutation in aparticular sub-unit.

In fact, the same beneficial effect was observed in the β- andδ-subunits, more precisely for the Q11E and E262K mutations respectivelyirrespective of the inhibitor used.

For this reason, this treatment may potentially be administered fordisorders related to all known mutations in the four human sarcoglycans.However, only point mutations should be considered for this approachsince truncated proteins caused by nonsense mutations or deletions willnot, in theory, be active even if not degraded.

The main point mutations reported at the present time are listed in thetable below. This list is not exhaustive.

Nucleotide Amino acid Exon substitution substitution α-sarcoglycan 02c.92T > C p.Leu31Pro 02 c.100C > T p.Arg34Cys 02 c.101G > A p.Arg34His03 c.184T > C p.Tyr62His 03 c.203G > A p.Gly68Gln 03 c.220C > Tp.Arg74Trp 03 c.229C > T p.Arg77Cys 03 c.266_267inv p.Leu89Pro 03c.269A > G p.Tyr90Cys 03 c.271G > C p.Gly91Arg 03 c.278C > T p.Ala93Val03 c.290A > G p.Asp97Gly 03 c.292C > T p.Arg98Cys 03 c.293G > Ap.Arg98His 03 c.308T > C p.Ile103Thr 04 c.329G > T p.Arg110Leu 04c.371T > C p.Ile124Thr 05 c.409G > A p.Glu137Lys 05 c.421C > Ap.Arg141Ser 05 c.472C > T p.Leu158Phe 05 c.518T > C p.Leu173Pro^(a) 05c.524T > C p.Val175Ala^(a) 05 c.541C > T p.Arg181Cys 06 c.586G > Ap.Val196Ile 06 c.614C > A p.Pro205His 06 c.662G > A p.Arg221His 06c.683C > A p.Pro228Gln 06 c.724G > T p.Val242Phe 06 c.725T > Cp.Val242Ala 06 c.739G > A p.Val247Met^(a) 07 c.850C > T p.Arg284Cysβ-sarcoglycan 01 c 31C > G p Gln11Glu 03 c.265G > A p.Val89Met 03c.271C > T p.Arg91Cys 03 c.272G > C p.Arg91Pro 03 c.274_275AT > TCp.Ile92Ser 03 c.275T > C p.Ile92Thr 03 c.299T > A p.Met100Lys 03c.323T > G p.Leu108Arg 03 c.341C > T p.Ser114Phe 03 c.355A > Tp.Ile119Phe 03 c.416G > A p.Gly139Asp 04 c.452C > G p.Thr151Arg 04c.499G > A p.Gly167Ser 04 c.538T > C p.Phe180Leu 04 c.544A > Gp.Thr182Ala 04 c.551A > G p.Tyr184Cys δ-sarcoglycan 04 c.212G > Cp.Arg71Thr 06 c.451T > G p.Ser151Ala 08 c.593G > C p.Arg198Pro 08c.631A > T p.Asn211Tyr 09 c.784C > A p.Glu262Lys γ-sarcoglycan 03c.205G > C p.Gly69Arg 03 c.206G > C p.Gly69Asp 03 c.269T > C p.Leu90Ser07 c.581T > C p.Leu194Ser 07 c.629A > G p.His210Arg 08 c.787G > Ap.Glu263Lys 08 c.848G > A p.Cys283Tyr

A second aspect of the invention relates to the assay used for in vitroassessment of the efficacy of inhibitors of the endoplasmic reticulumassociated degradation (ERAD) pathway, used for the treatment ofsarcoglycanopathies. This assay consists of the following steps:

-   -   co-transfection of cells with the four sarcoglycan genes (γ-,        α-, β- and δ-), of which one carries the mutation to be tested;    -   incubation for several hours in the presence of the inhibitor;    -   localisation of the sarcoglycan complex.

To advantage, human sarcoglycan genes carrying point mutations should beused.

If the complex is found to be located in the plasma membrane, then theinhibitor is identified as effective for the mutation being tested, andtherefore can be considered for use in a therapeutic approach aiming totreat that specific form of sarcoglycanopathy.

Detection of the complex in the plasma membrane implies that:

-   -   firstly the inhibitor has prevented degradation of the defective        protein;    -   and the mutant protein has translocated correctly and has        integrated the plasma membrane complex.

To advantage, this procedure is undertaken in parallel on:

-   -   cells co-transfected with the four wild-type sarcoglycan genes:        this is the positive control;    -   cells incubated in the same experimental conditions but without        the inhibitor: this is the negative control and provides the        basis for assessing the possible effects of the inhibitor.

Preferably, detection of the complex is performed byimmunohistochemistry, using antibodies against at least one of thesarcoglycans.

Alternatively, one of the sarcoglycans can be tagged, notably usingfluorescent tags, and then detected by microscopy or flow cytometry.This solution is advantageous compared to immunological techniquesbecause it involves fewer experimental steps. However, it is importantto check that the fusion protein (sarcoglycan+tag) does not disturbassembly of the complex.

In both cases, detection methods can, but do not necessarily, target thesarcoglycan carrying the mutation (choice of antibody or choice ofsarcoglycan to be tagged).

It should be noted that this method for screening inhibitors may be usedin vivo, in particular in transgenic mice bearing the relevantsarcoglycan mutation.

The invention and the advantages that it presents are best demonstratedby the following series of experiments illustrated by the figuresappended to this document. However, these experiments should not beconsidered to limit the scope of this invention.

The invention is mostly illustrated by experiments using anα-sarcoglycan protein carrying the R77C mutation in the presence ofMG132 (proteasome inhibitor) and kifunensin (mannosidase I inhibitor).

FIG. 1: Immunohistochemical labelling of α-, β-, γ-sarcoglycans anddystrophin on a muscle biopsy specimen from a patient carrying theα-sarcoglycan R77C mutation.

FIG. 2: Immunohistochemical labelling of α-, β-, γ-sarcoglycans anddystrophin in the mouse, in normal (WT), Sgca^(77C/77C) and Sgca−/−muscles.

FIG. 3: Histology of Sgca−/−, WT and Sgca^(77C/77C) muscles(Qua=quadriceps, Ga=gastrocnemius, Pso=psoas, Del=deltoid).

FIG. 4: Detection of necrotic cells using Evans blue staining in Sgca−/−and Sgca^(77C/77C) muscles.

FIG. 5: Immunohistochemical labelling of α-sarcoglycan and β-sarcoglycanin Sgca−/− muscles after injection of AAV-humanSgca77C. Left:α-sarcoglycan; centre: β-sarcoglycan; right: both α- and β-sarcoglycans.

FIG. 6: Histology of Sgca−/− muscles after injection of AAV-Sgca77C(left: non-transduced area; right: transduced area).

FIG. 7: Immunohistochemical labelling of α-sarcoglycan innon-permeabilised quadritransfected cells. Left: quadritransfection withnormal α-sarcoglycan; right: with mutant α-sarcoglycan.

FIG. 8: Immunohistochemical labelling of α-sarcoglycan (left) andcalreticulin (centre). Right: overlay of both stains.

FIG. 9: Immunohistochemical labelling of α-sarcoglycan inquadritransfected cells with mutant α-sarcoglycan in the presence ofMG132.

FIG. 10: Immunohistochemical labelling of α-sarcoglycan inquadritransfected cells with mutant α-sarcoglycan in the presence ofkifunensin.

FIG. 11: Immunohistochemical labelling of α-sarcoglycan showing theabsence of aggregates after a week of treatment with kifunensin in mouseSgca−/− muscle injected with mutant α-sarcoglycan.

FIG. 12: A/Immunohistochemical labelling of α-sarcoglycan in cellsquadritransfected with the three β-, γ- and δ-sarcoglycans and normalα-sarcoglycan (left), or mutant R77C α-sarcoglycan without (centre) orwith (right) 100 μM 1-deoxymannojirimycin (dMJ).

B/Immunohistochemical labelling of δ-sarcoglycan in cellsquadritransfected with the three α-, β- and γ-sarcoglycans and mutantE262K δ-sarcoglycan with or without 1-deoxymannojirimycin (dMJ) 5 or 100C/Immunohistochemical labelling of β-sarcoglycan in cellsquadritransfected with the three α-, γ- and δ-sarcoglycans and mutantQ11E β-sarcoglycan with or without 1-deoxymannojirimycin (dMJ) 5, 50 or100 μM.

FIG. 13: A/Immunohistochemical labelling of δ-sarcoglycan in cellsquadritransfected with the three α-, β- and γ-sarcoglycans and mutantE262K 6-sarcoglycan with or without kifunensin 5 μM.

B/Immunohistochemical labelling of β-sarcoglycan in cellsquadritransfected with the three α-, γ- and δ-sarcoglycans and mutantQ11E β-sarcoglycan with or without kifunensin 5 μM.

I) MATERIAL AND METHODS

1-Production of Sgca^(77C/77C) Mice

A 1075 bp DNA fragment carrying exons 2 and 3 of the Sgca gene obtainedby BamHI-SfiI digestion of a phage containing the Sgca gene wasamplified by PCR and then inserted into the pSP72 plasmid vector(Promega). The H77C mutation was generated in exon 3 by site-directedmutagenesis using the following primers:5′-GCCCAGGTGGCTGTGCTACACACAGCGCA-3′ (SEQ ID 1) and5′-TGCGCTGTGTGTAGCACAGCCACCTGGGC-3′ (SEQ ID 2). Presence of the pointmutation was confirmed by sequencing. A 5′ BglII-BamHI fragment of about4 kb and a 2981 bp 3′ SfiI-SpeI fragment were cloned into the pSP72vector on each side of the mutant insert. The loxP-neo^(r)-loxP cassettefrom the pGEM-neo^(r) vector was inserted via the EcoRV site in intron 3and a thymidine kinase (TK) cassette was inserted downstream of the 3′fragment to produce the final recombinant vector.

The recombinant vector (25 μg) was linearised by SalI digestion andintroduced into SE 129Sv cells by electroporation. The DNA of G418resistant colonies was then isolated and analysed by PCR or Southernblot to check for recombination events. Two distinct recombinant clones(IB4 and VIIICII) were injected into C57B1/6 blastocysts and chimeramice were generated. Chimeric males were crossed with C57B1/6 females toproduce heterozygous mice. The neo^(r) cassette was eliminated bycrosses with the deleter strain (15). The resulting mice in which theneo^(r) cassette had been excised were then bred to produce homozygousmutant mice.

Genotyping was performed by PCR on tail DNA extracted using the QiagenDNeasy Tissue Kit, using the upstream primers a-sarcoQ5′:5′-TATAACCCTGGCTTCCTCTA-3′ (SEQ ID 3) and testNeo 15′-CGAATTCGCCAATGACAAGACGCT-3′ (SEQ ID 4) and the downstream primera-sarcoQ3′ 5′-TAGTGGCTCATGCCTTTAAT-3′ (SEQ ID 5), generating a 639 bpproduct for the mutant allele carrying the neo^(r) cassette and a 484 bpproduct for the wild-type allele, using the following PCR conditions:94° C. for 3 minutes, then 30 cycles consisting of 94° C. for 30 s, 61°C. for 40 s and 72° C. for 1 minute, and then 3 minutes at 72° C. Afterexcision of the neo^(r) cassette, genotyping was performed with thea-sarcoQ5′ (SEQ ID 3) and a-sarcoQ3′ (SEQ ID 5) primers, generating a575 bp product for the mutant allele.

To check that the H77C mutation was present in the Sgca gene of themodel, a PCR was performed on tail DNA with primers KIgenoseq2.s5′-TGTGTTTGGGACTTATGGGG-3′ (SEQ ID 6) and KIgenoseq2.as5′-CAATCAGCAGCAGCAGCCTC-3′ (SEQ ID 7) generating a 659 bp PCR productthat was then sequenced.

2-Histology and Immunohistochemistry

8 μm cross-sections from frozen muscle were stained using haematoxylinand eosin (H&E). Cross-sections from mice injected with Evans blue wererevealed by fluorescent excitation at 633 nm.

Cross-sections were dried, then rehydrated in PBS or fixed cells weretreated for 20 min with 1% triton in PBS, then incubated for 30 min atroom temperature (RT) in PBS containing 15% foetal calf serum.Cross-sections were incubated with polyclonal anti-sarcoglycanantibodies (α-sarcoglycan: dilution 1/1000, targeting amino acids366-379 of human α-sarcoglycan; β-sarcoglycan: dilution 1/20, NCL-b-SARC(Novocastra); γ-sarcoglycan: dilution 1/20, NCL-g-SARC (Novocastra);dystrophin: dilution 1/20, NCL-DYS2 (Novocastra); and calreticulin:dilution 1/70, ab4109 (Abcam)) for 1 to 2 hours at RT then rinsed 3times in PBS. Primary antibodies were revealed after 1 hour incubationat RT with secondary antibodies conjugated with fluorochromes A1exa488(A-11032, Molecular Probes) or A1exa594 (A-11037, Molecular Probes),diluted 1:1000 in PBS. Cross-sections were then rinsed three times inPBS, mounted with fluoromount-G (Southern Biotech 0100-01) and thenobserved with a confocal microscope (Leica). Immunohistochemicalanalysis of human biopsy specimens was performed as described in Hackmanet al. (9).

Plasmids pAAV_C5-12_α-SG, pcDNA3_α-SG, pcDNA3_β-SG, pcDNA3_γ-SG andpcDNA3_δ-SG were produced by PCRs on skeletal muscle cDNA and clonesusing the TOPO TA cloning kit (Invitrogen). Sarcoglycans were thensubcloned into the pcDNA3 plasmid (Invitrogen) or pAAV_C5-12_MCS(3).Plasmids pcDNA3_α-SG-R77C and pAAV_C5-12_α-SG-R77C were obtained frompcDNA3-α-SG or pAAV_C5-12_α-SG by site-directed mutagenesis using theQuickchange site-directed mutagenesis kit (Stratagene) and the followingprimer: 5′-GCCCCGGTGGCTCTGCTACACCCAGCGC-3′ (SEQ ID 8). The constructionswere checked by enzymatic digestion and sequencing.

The adenovirus-free AAV 2/1 viral preparations were produced byintroducing recombinant AAV2-ITR genomes into AAV1 capsids using thetritransfection protocol (2). Viral genomes were quantified by dot blotin comparison with a series of standard plasmid dilutions.

4-Cell Culture

NIH3T3 or 911 cells were grown in Dulbecco's Modified Eagle Mediumsupplemented with glutamine, gentamicin and 10% foetal calf serum. Cellswere transfected using 6 μA Fugene (ROCHE) per 1 μg plasmid. 0.5 μg ofeach plasmid (α-SG or α-SG-R77C and β-SG, γ-SG, δ-SG) was used per wellin 6-well dishes. For treatment by inhibitors, 43 hours aftertransfection cells were incubated for 5 hours with either themannosidase inhibitor (kifunensin 5 μM, VWR) or the proteasome inhibitor(MG132 5 μM diluted in DMSO, Sigma). Cells were then rinsed in PBS andfixed with 3.7% formaldehyde in PBS for 15 min at RT, and rinsed threemore times in PBS before immunohistochemical labelling.

5-In Vivo Experiments

Sgca^(77C/77C) mice were exercised for 30 minutes per day on a treadmill(Columbus treadmill instrument Exer 6M) on 3 consecutive days. Mice wereplaced on a treadmill with a downward incline of 15° and the speed wasset at 10 metres per minute. At the end of the 3 days, mice wereinjected intraperitoneally with Evans blue dye (0.5 mg/g). Mice weresacrificed the day following injection and the deltoid, psoas,gastrocnemius, gluteus, extensor digitorum longus and quadriceps muscleswere dissected and frozen rapidly in isopentane chilled with liquidnitrogen.

The rAAV2/1 viral preparations were injected (10¹⁰ viral genomes (vg) in30 μl total volume) in the left tibialis anterior muscle of Sgca−/−mice. On days 20, 22, 25 and 27 after injection, 10 μM kifunensin or 20μM MG132 were injected into the muscle (i.e. either twice or 4 times theconcentrations used in vitro to take into account diffusion within themuscle). One day prior to sacrifice (day 27), mice were injectedintraperitoneally with Evans blue dye. Both left and right tibialismuscles were dissected and frozen rapidly in isopentane chilled withliquid nitrogen.

II) RESULTS

1-Mutant R77C α-sarcoglycan is Absent from the Membrane in Humans.

The presence of mutation R77C in α-sarcoglycan in humans leads todestabilisation of the sarcoglycan complex, as shown byimmunohistochemical labelling using antibodies against various distinctproteins of the complex (FIG. 1).

2-In the Mouse, the Presence of a Cysteine in Position 77 Does notPrevent α-sarcoglycan Membrane Targeting and Does not Result in aPathological Condition.

In order to investigate the reasons for complex destabilisation, weproduced, by homologous recombination, an animal model (Sgca^(77C/77C))bearing a cysteine in position 77. It should be noted that normal micebear a histidine residue at this position and not an arginine residue.Immunohistochemical analysis of muscles from these mice using antibodiesagainst various DGC complex proteins demonstrated that this mutation didnot prevent α-sarcoglycan membrane targeting or assembly of thesarcoglycan complex (FIG. 2). Tissue cross-sections from wild-type andα-sarcoglycan deficient (Sgca−/−, 8) mice were used as controls.

The histology of the deltoid, psoas, gastrocnemius and quadricepsmuscles from 3 to 6 month old SgCa^(77C/77C) mice was examined byhaematoxylin/eosin staining and compared to Sgca−/− mouse tissue.Although the latter display the signs of severe dystrophy, no anomalieswere detected in Sgca^(77C/77C) muscles (FIG. 3).

The absence of muscle anomalies was confirmed by functional analysis.SgCa^(77C/77C) mice were subjected to exercise enhancing eccentricmuscle contractility and then injected intraperitoneally with Evans bluedye, a dye that specifically stains necrotic cells. No Evans blue dyepenetration was observed in the muscles of SgCa^(77C/77C) mice (FIG. 4).

To determine whether the differences observed between mice and humanswhen a cysteine residue is present in position 77 were related to theintrinsic properties of the human protein, we performed gene transferexperiments in muscles of α-sarcoglycan deficient mice using a viralvector derived from the adeno-associated virus (AAV) carrying the humanα-sarcoglycan gene mutated in position 77. Analysis of these injectedmuscles demonstrated that the mutant protein is localised in themembrane, although some protein is retained in the reticulum, that thesarcoglycan complex assembles and that the pathological phenotype iscorrected (FIGS. 5 and 6). It should be noted, as seen in FIG. 5, thatα-sarcoglycan accumulates in the perinuclear space in some cells.

3-The Mutation Causes α-sarcoglycan Retention and its Degradation by theProteasome.

We have established a cellular model reproducing the phenomena observedin humans by quadritransfecting plasmids coding the four differentsarcoglycans. In this model, the complex assembles correctly at themembrane when normal α-sarcoglycan is co-transfected together with thethree other sarcoglycans. However, correct assembly is not observed atthe membrane when the R77C α-sarcoglycan is transfected. This wasdemonstrated by immunohistochemical labelling using an antibody againstthe extracellular segment of α-sarcoglycan on non-permeabilised cells(FIG. 7).

Double-labelling of α-sarcoglycan and an endoplasmic reticulum marker(calreticulum) in permeabilised cells showed that mutant protein wasretained in the secretion pathway (FIG. 8).

We postulated that mutant α-sarcoglycan was recognised as abnormal bythe protein quality control system of the reticulum and then degraded bythe proteasome. This was confirmed by use of the proteasome inhibitorMG132 which restored membrane targeting (FIG. 9).

The protein quality control system of the reticulum leads to degradationof incorrectly folded proteins. The mannosidase I enzyme plays animportant role in this process by modifying the oligosaccharide chainsof glycosylated proteins such as sarcoglycans. Bearing in mind thesefacts and our results, we postulated that use of an inhibitor of thisenzyme might restore membrane targeting of the mutant α-sarcoglycanprotein. This hypothesis was validated in our cellular model in cellsquadritransfected with mutant α-sarcoglycan and then treated bykifunensin (FIG. 10).

The differences observed between humans and mice meant that we did nothave an in vivo mouse model corresponding to the molecular eventsobserved in humans. However, a decrease in the partial retention of themutant protein after gene transfer would suggest that mannosidase Iinhibition could have a beneficial effect on α-sarcoglycan membranetargeting. To determine whether use of the inhibitor might be effectivein vivo, Sgca−/− mice first received an injection of the AAV-SGCA77Cvector and then (15 days later) received three intramuscular injectionsof the inhibitor over a one week period. Muscles that were injected withthe inhibitor displayed a nearly complete absence of intracellularaggregates, and notably an absence of accumulation in the perinuclearspace. These results validated our working hypothesis (FIG. 11).

The same convincing results were obtained with kifunensin for two otherpoint mutations on two other sub-units from the sarcoglycan complex:mutation E262K on sub-unit δ (FIG. 13A) and mutation Q11E on sub-unit β(FIG. 13B).

5-Restoration by 1-deoxymannojirimycin (dMJ)

Experiments similar to those performed with kifunensin on mutant R77Cα-sarcoglycan were performed using 1-deoxymannojirimycin (dMJ), anotherclass I mannosidase inhibitor. Results are shown in FIG. 12A anddemonstrate the efficacy of this substance for restoring membranetargeting of mutant α-sarcoglycan (R77C mutation).

In addition, similar experiments confirmed our working hypothesis forother mutations on other sub-units of the sarcoglycan complex: mutationE262K on sub-unit δ (FIG. 12B) and mutation Q11E on sub-unit β (FIG.12C).

To conclude, we have shown that mutant α-sarcoglycan bearing a cysteinein position 77 is managed quite differently in mouse and human cells,and that this mutant protein is functional if it is located correctlyand can remedy the pathological condition related to sarcoglycandeficiency. We have demonstrated, in a cellular model, that mannosidaseI inhibition prevents degradation of mutant sarcoglycan and restoresmembrane targeting. Use of this substance in vivo also seems to producethe same results.

REFERENCES

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1-10. (canceled)
 11. A method of treating a sarcoglycanopathy comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount of a class I α-mannosidase inhibitor.
 12. A methodaccording to claim 11 wherein said inhibitor comprises a molecule thathas class I α-mannosidase inhibitory activity.
 13. A method according toclaim 12 wherein said inhibitor comprises kifunensin or1-deoxymannojirimycin.
 14. A method according to claim 13 wherein saidinhibitor comprises kifunensin.
 15. A method according to claim 11wherein said sarcoglycanopathy is associated with the R77C mutation onhuman α-sarcoglycan.
 16. A method according to claim 11 wherein saidsarcoglycanopathy is associated with the E262K mutation on humanδ-sarcoglycan.
 17. A method according to claim 11 wherein saidsarcoglycanopathy is associated with the Q11E mutation on humanβ-sarcoglycan.
 18. A method for assessing the efficacy of class Iα-mannosidase inhibitors on a sarcoglycanopathy comprising the followingsteps: co-transfecting cells with the four sarcoglycan genes (γ-, α-, β-and δ-), of which one carries a mutation; incubating for several hoursin presence of an inhibitor according to claim 11; determining thelocalisation of the sarcoglycan complex within the cell; and assessingthe efficacy on the basis of said localisation.
 19. A method forassessing inhibitor efficacy according to claim 18 wherein said methodis carried out in parallel on cells co-transfected with the genes codingfor the four wild-type sarcoglycans.
 20. A method for assessinginhibitor efficacy according to claim 18 wherein said method is carriedout in parallel on cells incubated without the inhibitor.
 21. A methodfor assessing inhibitor efficacy according to claim 18 furthercomprising the use of immunohistochemical labelling techniques forlocalising the complex.